EFFECT OF RESTRICTED FEEDING LEVEL ANDWATER RENEWAL ON WATER QUALITY AND NILE

TILAPIA GROWTH PERFORMANCE UNDER BIOFLOCCULTURE

By

AML FARAG YOUNESB.Sc. Agric. Sci. (Animal Production), Fac. Agric., Omar Almukhtar Univ., Libya, 2008

THESISSubmitted in Partial Fulfillment of the

Requirements for the Degree of

MASTER OF SCIENCE

In

Agricultural Sciences(Fish Production)

Department of Animal ProductionFaculty of Agriculture

Cairo UniversityEGYPT

2015

نمو البلطي ید المیاه علي جودة المیاه وأداءالمحدوده وتجدتأثیر مستوي التغذیھ النیلي تحت زراعة البیوفلوك

رسالة مقدمة من

أمل فرج یونس2008لیبیا، ،عمر المختارجامعة ،كلیة الزراعة،)انتاج حیوانيالعلوم الزراعیة (يفبكالوریوس

درجةللحصول على

الماجستیر

في

الزراعیةالعلوم )انتاج االسماك(

النتاج الحیوانيقســـــــم اكلیــــــة الزراعـــــةجـامعــة القــاھرة

ــــرمصــــ

2015

.فاعلیة اإلنتاج-البلطى النیلى- تجدید المیاه- التغذیة المحدوده-البیوفلوك الكلمات الدالة:

ABSTRACTThe current experiment investigated the effect of restricted feeding and partial water renewal in

biofloc tank culture on growth and feed performance of Nile tilapia. The design of the experimentincluded four biofloc treatments, with two feeding levels (75 and 100 grams diet /m3/day) and twopartial water renewal rates (weekly and biweekly). The control treatment was fed to satiation, withwater renewal each other day. In spite of the differences in growth rates, the higher protein efficiencyratios (2.14- 2.59) and the better food conversion ratios (1.21 – 1.76 :1 ) observed in the biofloctreatments indicated that it is possible to use restricted feeding when fish are raised in biofloc tanks.The biofloc treatments were more efficient in terms of feed conversion ratios and survival. rateConsequently, the current results suggest that in the presence of biofloc, it is possible to restrict(reduce) feeding rates from satiation to restricted feeding without affecting harvest volume or survivalrate. The modified biofloc systems could contribute to the nutrition and physiological health of Niletilapia.. The results indicated that the rested biofloc treatments were more efficient in terms of FCR andPER values due to feed restriction to 75 to 100 g diet /m3/day. The control treatment which was fed tosatiation at 125 g diet/m3/day produced better final body weight and daily weight gain with less feedefficiency, reduced survival rates and higher pumping costs.Keywords: Biofloc , Restricted Feeding , Water Renewal , Nile tilapia , Efficiency,

Production.

DEDICATION

Dedicate this work to be stopped next to me and wasa beacon lights intellectual maturity and guidance in oldage and the full tender me the confidence to continue tocomplete their higher Mrahali Scholastic my dear fatherand to whom Helena compassion and tenderness and that

المستخلص العربياھتمت التجربھ الحالیھ بدراسة تاثیر التغذیھ المحدوده بنظام البیوفلوك والتجدید الجزئى للمیاه في احواض تربیة

اداء النمو وكفاءة التغذیھ للبلطي النیلي .وقد اشتمل تصمیم التجربھ على اربعة معامالت بیوفلوك االسماك علي كل - /یوم) ومستوین من التجدید الجزئى للمیاه (اسبوعیا3جرام علیقھ /م100-75تستخدم مستویین من التغذیھ (

اع مع تجدید المیاه كل یومین. بالنسبھ اسبوعین). اما تغذیة االسماك في معاملة الكنترول كانت عند حد االشبجرام 67.9-55.8لوزن الحصاد في اسماك البلطي اظھرت التجربھ ان وزن الحصاد في معامالت البیوفلوك (

جرام /سمكھ).وبالرغم من ذلك كان الوزن الكلي للحصاد في 91.4/سمكھ)كانت اقل معنویا من معاملة الكنترول(كیلو جرام اسماك/ متر مكعب في معامالت 6.08-5.15حیث كان یتراوح بین جمیع المعامالت متساوي معنویا

البیوفلوك ومعاملة الكنترول .وھذا یرجع الي ان نسبة الحیاه كانت مرتفعھ في معامالت البیوفلوك وكانت - 2.14تین (منخفضھ في معاملة الكنترول .وایضا تمیزت معامالت البیوفلوك بالكفاءه العالیھ في نسبة كفاءة البرو

). ولذلك توضح التجربھ انھ من الممكن ان نستخدم 1:76,1- 1,21) ومعدل التحویل الغذائي المثالي (2.59التغذیھ المحدوده عند تربیة اسماك البلطي النیلي في نظام البیوفلوك وقد كانت معامالت البیوفلوك ذات كفاءه

بروتین ونسبة الحیاه في االحواض .لذلك توصي الدراسھ عالیھ من ناحیة معدل التحویل الغذائي ونسبة كفاءة الالحالیھ بانھ من الممكن تقلیل معدل تغذیھ البلطي النیلي من مستوي االشباع الي التغذیھ المحدوده بدون التاثیر على حجم المحصول او نسبة الحیاه او معدل التحویل الغذائي .ویساھم وجود البیوفلوك في میاه االحواض في

ة البلطي النیلي والصحھ الفسیولوجیھ لالسماك.تغذی

which has relevance to him, thanks to the continuouspraying to my mother Higher & Dedicate well as this workto my sisters and sisters and to all my friends in Libya.

ACKNOWLEDGEMENT

If gifting crosses part of the fulfillment of Validate to theteacher of mankind and the source of the flag of our ProphetMuhammad peace be upon him and to whom and that I should extendmy deep thanks and gratitude and sincere gratitude to all who directedme and took my hand in order to accomplish this letter and morespecifically Belcher D / Mohammed ELNADY Ahmed, the people andtowards He continued

As aspects of gratitude and respect to Dr. / Gamal Ashour, headof the Department of Animal Production and specifically Blchukr andappreciation to my entire friends Raffia path that Cano Mai in everystep of the Scholastic Marshal

As geld thanks and appreciation to the company of Derby andmy sister, and that was with me Tilt Scholastic period for its help in allrespects, particularly Blchukr and appreciation Professor/ Heba JamalRahuan Whatley and stood beside me even made the dream come trueReally I do not want forget any one supporting Egypt my secondfamily home and my country.

INTRODUCTION

Aquaculture is predicted to increase 5-folds by 2050 (FAO, 2012).

To meet this growing demand, aquaculture is shifting from extensive

cultivation systems to more intensive systems (Luo et al., 2013). With the

intensification of aquaculture, the focus has increasingly shifted to its

negative environmental and social impact (Luo et al., 2013). Even RAS,

which are considered to provide more advantages than traditional

aquaculture, have also been reported to accumulate 11–40% of the

applied feed in the form of discharged sludge (Hopkins et al., 1994).

Aqua culturists continue to increase their interest in and use of

mixed suspended-growth production systems, also referred to as biofloc

technology (BFT) systems, for culturing various aquatic animal (Schrader

et al., 2011). These BFT systems rely on the living microorganisms in the

biofloc (composed of microbial biomass and particulate organic matter)

maintained in the water column to assist in ammonia removal via

phytoplankton and bacterial uptake (Schrader et al., 2011) and bacterial

oxidation of ammonia-N (NH3-N) to nitrite-N (NO2-N) and then

Subsequent oxidation of NO2-N to nitrate-N (NO3-N) during nitrification

(Brune et al., 2003; Ebeling et al., 2006 and Hargreaves, 2006).

Biofloc is composed of bacteria, fungi and plankton that have

higher protein content (30-40%). The size of biofloc particles ranges from

0.5 to 2.0 mm, which could be fed to Nile tilapia and shrimp (Supono et

al., 2013).

Biofloc culture could utilize heterotrophic bacteria to convert

ammonia produced in aquaculture into bacterial biomass (De Schryver

and Verstraete, 2009), which could potentially be used to feed fish,

thereby increasing feed efficiency (Luo et al., 2013). Nile tilapia and

heterotrophic bacterial biomass are cultured in the same water volume

and has already been exploited in pond aquaculture systems for tilapia

(Azim and little, 2008 and Crab et al., 2009).

In biofloc technology (BFT) the growth of heterotrophic bacterial

biomass is stimulated towards the conversion of the excreted ammonia

waste into microbial biomass by supplementing an external carbohydrate

source (i.e. molasses or sucrose). This biomass can be further used as a

food source by the cultured organisms, therefore increasing feed

utilization efficiency (Ekasari et al., 2013). Therefore, these biological

processes play a critical role in reducing ammonia and nitrite to levels

below those that can be toxic or growth-limiting for cultured finfish

(Schrader et al., 2011).

As feed is the major driving force of intensive production

systeams, it is important to optimize its use to improve profitability,

maximize growth, and minimize potential water quality deterioration

(Correia et al., 2014). Biofloc rearing media provides a potential food

source for shrimp reared in limited or zero water exchange systems. This

culture system is environmentally friendly as it is based on limited water

use and minimal effluent is released into the surrounding environment

(Emerenciano et al., 2011).

Freshwater scarcity is for sure becoming global concern due to

high growth rate of human population. Use of biofloc technology

encourages water conservation. Ogello et al. (2014) indicated that

significant reduction in organic nitrogen accumulation, increased

utilization of feed protein and reduced feed expenditure in biofloc

systems.

Intensive, recirculating tilapia, Oreochromis niloticus bioflocsystems are capable of producing the equivalent of 155 tons /ha/crop(Rakocy et al., 2004). Biofloc production systems treat and reuse a majorportion of their water, but depend on the discharge of nitrogenous wastesand organic matter to ensure system sustainability (Danaher et al., 2011).

The aims of the present study were to evaluate growth performanceand dietary efficiency for Nile tilapia cultured in intensive biofloc systemusing different feeding levels. The objective was to evaluate the bestbiofloc system under restricted feeding and partial water renewalconditions compared to clear water culture conditions. The study hadthree major objectives: (1) to determine the effect of different feedingrates at 75.0, 100.0 and 125.0 grams diet/m3/day on tilapia growth,survival rare, feed efficiency and selected water quality parameters indifferent biofloc systems, (2) to determine if sucrose can be used toprevent ammonia and nitrite accumulation, and (3) to determine the effectof weekly and biweekly partial water renewal on growth and waterquality parameters.

REVIEW OF LITERATURE

1. Biofloc advantages

Biofloc technology (BFT) has recently gained great attention as a

sustainable solution that not only can effectively control water quality under

zero-water exchange but also sustain intensive and healthy culture of shrimp

(Crab et al., 2012; Stokstad and Taw ,2010). The driving force of BFT culture

systems is microbial biofloc, which is a heterogeneous aggregate of suspended

organic particles and many varieties of active microorganisms associated with

extracellular polymeric substances (De Schryver et al., 2008; Jue et al., 2008b;

Ray et al., 2010).

The biofloc has been reported to confer many beneficial effect on

shrimp culture ( Xu and Pan 2013) including: (1) improving water quality

through removal of toxic nitrogen compounds such as ammonia and nitrite (De

Schryver et al., 2008; Ray et al., 2011and Xu et al., 2012); (2) increasing feed

utilization and growth performance of shrimp through supplementing natural

food and stimulating digestive enzyme activities (Ballester et al., 2010;

Emerenciano et al., 2012; Xu and Pan, 2012; Xu et al., 2012b); and (3)

enhancing biosecurity and health management through zero-water exchange

and possible probiotic effect (Crab et al., 2010; Haslun et al. , 2012 and Moss

et al.,2012 and Zhao et al., 2012).

BFT has been sought as a means of enhancing water quality through

microbial manipulation, thereby facilitating the growth and health of cultured

shrimp. In BFT zero-water exchange systems, carbohydrate addition can

promote the development of diverse and balanced microbial communities

originating from the rearing water (Haslun et al., 2012). These active and dense

microorganisms together with suspended organic particles tend to form biofloc,

which can be consumed constantly by cultured shrimp as a natural food source

(Buford et al., 2004; Kent et al., 2011 and Wasielesky et al., 2006).

Interest in super intensive shrimp culture with minimal or no water

exchange in biofloc technology (BFT) systems have emerged (Wasielesky et

al., 2013). In BFT system, highly oxygenated ponds are fertilized with carbon-

rich sources to stimulate heterotrophic bacterial biota (Ebeling al., 2006, De

Shriver and Verstraete 2009). The bacteria that inhabit bioflocs assimilate the

dissolved nitrogen compounds in the water, which are generated primarily by

shrimp excretion and the decomposition of organic matter (Crab et al. 2007 and

De Shryver et al. 2008).

This aquaculture practice enables the recycling of the culture water

through several cycles, making the system environmentally friendly

Krummenauer et al. 2012).

Furthermore, the stimulation provided by the bioflocs can be an

important feed supplement in the shrimp diet ( Wasielesky et al.,2013)

contributing to digestion and protein retention, and the scope of these benefits

includes the nursery phase (Tacon et al., 2002; Cuzon et al., 2004; Otoshi et al.,

2011 and Xu et al., 2012). The systems called BFT (Bio-floc technology) with

zero water exchange reduce not only the water use, but also the issuance of

effluent into the environment, avoiding the environmental damage (Burford et

al., 2003).

The most promising features of BFT systems (zero water exchange) are

that they increase biosecurity (Bullis and Pruder, 1999), reduce feed costs and

water use (Chamberlain and Hopkins, 1994; Boyd, 2000). In these systems, the

manipulation of the C/N ratio by the addition of carbohydrate significantly

reduced inorganic N concentrations in the water column and total nitrogen in

the sediment (Azim and Little, 2008).

At high carbon and nitrogen (C/N) ratio, heterotrophic microorganisms

dominate autotrophic microorganisms and assimilate total ammonia nitrogen,

nitrite and nitrate to produce cellular proteins that can serve as a supplemental

feed source for shrimp (Stokes and McIntosh, 2001; Buford and Lorene,

2004), making it a low-cost sustainable constituent to future aquaculture

development (De Shryver et al., 2008).

Conventional technologies to manage and remove nitrogen compounds

are based on either earthen treatment systems or a combination of solids

removal and nitrification reactors (Crab et al., 2007). These methods have the

disadvantage of requiring frequent maintenance and in most instances the units

can achieve only partial water purification (Crab et al., 2012).

Biofloc technology, on the other hand, is robust, economical technique

and easy in operation. One important aspect of the technology to consider is the

high concentration of total suspended solids present in the pond water. Suitable

aeration and mixing needs to be sustained in order to keep particles in

suspension and intervention through either water exchanger or drainage of

sludge might be needed when suspended solids concentrations become too high

(Crab et al., 2012).

Although it is a critical aspect of biofloc technology, detailed

knowledge about selection and placement of aerators is still lacking (Crab et

al., 2012). Future research should address this issue and could also investigate

new concepts, such as the integration of biofloc technology in raceways.

Construction aspects for biofloc technology ponds merely deal with aeration.

When establishing biofloc technology in aquaculture ponds, a certain

start-up period is needed to obtain a well-functioning system with respect to

controlling water quality and this will depend on the nitrogen and organic load

of the culture water and thus the intensity of the system (Crab et al., (2012).

However, because heterotrophs grow at a rate that is 10 times higher than that

of nitrifying bacteria in biofilters (Crab et al., 2007), bioflocs can usually be

established much faster than conventional bio filters (Crab et al., 2012).

Translated in biofloc terms, ‘waste’-nitrogen generated by uneaten feed

and excreta from the cultured organisms is converted into proteinaceous feed

available for those same organisms. Instead of ‘down cycling’, a phenomenon

often found in an attempt to recycle, the technique actually ‘up cycles’ through

closing the nutrient loop (Crab et al., 2012). Hence, the water exchange can be

decreased without deterioration of water quality and, consequently, the total

amount of nutrients discharged into adjacent water bodies may be decreased

(Lezama-Cervantes and Paniagua-Michel, 2010).

In this context, biofloc technology can also be used in the specific case

of maintaining appropriate water temperature, good water quality and high fish

survival in low/no water exchange, greenhouse ponds to overcome periods of

lower temperature during winter (Crab et al ., 2012). Indeed, fish survival

levels in overwintering tilapia cultured in greenhouse ponds with biofloc

technology were excellent, being 97% for 100 g fish and 80% for 50 g fish

(Crab et al., 2009). Moreover, at harvest, the condition of the fish was good in

all ponds.

Decreased water exchange reduces pollutant discharge, disease

exchange between wild and captive stocks, and introductions of exotic species

to the wild (Ray et al., 2011). The microbial community in intensive minimal-

exchange culture systems is responsible for cycling nutrients most importantly

nitrogen compounds (Ray et al., 2011) Feed decomposition and animal

excretions contribute to ammonia which is toxic to shrimp (Ray et al., 2011).

Algae and heterotrophic bacteria can directly assimilate ammonia to build

cellular proteins, and nitrifying bacteria can oxidize ammonia to form nitrite

and nitrate (Ebeling et al., 2006). Each of these three groups contribute to

detoxifying nitrogenous waste, but each has drawbacks i.e. algae are limited in

the amount of nitrogen they can remediate (Brune et al., 2003).

2. Biofloc composition

Bioflocs sampled from biofloc-based tanks were observed as brown in

color, ranging in size from 0.2 to 4 mm. The bioflocs were composed of

suspended organic particles in the form of flocculated aggregates, which were

colonized by a number of heterotrophic bacteria, microalgae and protozoa (Xu

and Pan, 2013).

Suspended growth in ponds consists of phytoplankton, bacteria, aggregates

of living and dead particulate organic matter, and grazers of the bacteria

(Hargreaves, 2006). Typical flocs are irregular by shape, have a broad

distribution of particle size, and are fine, easily compressible, and highly

porous (up to more than 99% porosity) and are permeable to fluids (Chu and

Lee, 2004).

3. Carbon: nitrogen ratio

In such systems, a high C/N ratio (10:1 to 20:1) of feed input is

recommended for the establishment and development of biofloc (Asaduzzaman

et al., 2010; Hargreaves, 2006). In practice, adding carbohydrates (e.g. sucrose)

to the culture water as a supplement to the shrimp feed is an effective means to

increase the C/N ratio (Ebeling et al., 2006). This result suggests that it's

possible to manipulate an appropriately high C/N ratio of feed input through

carbohydrate addition to achieve a well-performing BFT system, and has a

positive application prospect in large-scale shrimp aquaculture (Xu and Pan,

2013).

Recently, manipulation of carbon nitrogen ratio (C: N ratio) for

development of biofloc has shown promise in aquaculture (Anand et al.,

2013a). In an experiment, C: N ratio was manipulated by supplementation of

external carbon source or elevated carbon level in the feed (Ballester et al.,

2010; McIntosh, 2000). At high C: N ratio, heterotrophic bacteria immobilize

the ammonium ion for production of microbial protein and maintain inorganic

nitrogen level within the limit.

Wheat flour was used as carbohydrate source for its easy availability

and production of good quality flock (Azim and little, 2008; Ballester et al.,

2010). In an experiment, 4.03 kilograms of wheat flour was used to produce 1

kg of microbial flocs (Shyne Anand et al., 2014). Earlier, Kuhn et al. (2009)

reported 1 kg microbial flocs production from 1.5 kg of sucrose in a bioreactor.

This may be because being a disaccharide, sucrose is readily available for

microbial utilization, while wheat flour has complex long chain

polysaccharides (Shyne Anand et al., 2014).

Even though, bioreactors have better conversion efficiency, the present

production system is simple and advantageous as it used cheap and readily

available ammonium sulphate as nitrogen source and wheat flour as carbon

source.(Shyne Anand et al., 2014).

If carbon and nitrogen are well balanced in the solution, ammonium, in

addition to organic nitrogenous waste, will be converted into bacterial biomass

(Schneider et al., 2005). Biofloc technology is a technique of enhancing water

quality through the addition of extra carbon to the aquaculture system, through

an external carbon source or elevated carbon content of the feed (Crab et al.,

2012). This promoted nitrogen uptake by bacterial growth decreased the

ammonium concentration more rapidly than nitrification (Hargreaves, 2006).

Immobilization of ammonium by heterotrophic bacteria occurs much

more rapidly because the growth rate and microbial biomass yield per unit

substrate of heterotrophs are a factor 10 higher than that of nitrifying bacteria

(Hargreaves, 2006). The microbial biomass yield per unit substrate of

heterotrophic bacteria is about 0.5 g biomass C/g substrate C used (Eding et al.,

2006). Downstream carbonaceous byproducts of local industry can provide low

cost external carbon source for application in biofloc technology in nearby

ponds, but will need preceding research before implementation.

Balancing the carbon content of the feed fed to the culture organism

could be an alternative to elevating the organic carbon to nitrogen ratio through

addition of an external organic carbon source (Crab et al., 2009). Crab (2010)

showed that with L. vannamei, bioflocs grown on glucose lacked accessibility

and palatability for good survival and growth. The latter opens an interesting

field of research, where one can look at carbon sources that would increase

attractiveness of the bioflocs toward fish and shrimp. A worthy carbon source

to look at in this regard is molasses obtained during sugar processing of sugar

beet (Beta vulgaris L.), which contains glycine betaine, a known attractants

used in aquaculture (Felix and Sahdaran, 2004; Macula et al., 1998).

Amount of organic supplementation was calculated based on the

methods of Ebeling et al. (2006), assuming that 6 g of carbon is needed to

convert 1 g of TAN (total ammonia nitrogen), generated from feed, into

bacterial biomass. Therefore, when the ammonia concentration in the

experimental tanks of the molasses treatment reached values of 1 mg/L or

higher, these tanks received a molasses dose calculated according to the

equations proposed by, Ebeling et al.2006.

Samocha et al. (2007) tested the molasses as a carbon source for shrimp

( L. vannamei) and demonstrated that the use of molasses resulted in

stimulation of heterotrophic bacterial floc formation that successfully competed

with the algal population in this environment with an augmented carbon

concentration.

In addition, the low levels of TAN and nitrite suggest that molasses

addition was an effective tool in controlling these nitrogen compounds (Souga

et al, 2012). Gao-Shan et al. (2012) tested sucrose as a carbohydrate to increase

C/N ratio to evaluate the water quality improvement and shrimp performance.

These authors observed that the concentrations of TAN and NO2 were kept at

significantly lower levels with the addition of a certain quantity of sucrose

(75% and 100%). Their results indicated that 75% and 100% could effectively

increase the C/N ratios of the water. Furthermore, they suggested that 75% of

the added quantity of may be appropriate for the L. vannamei intensive culture

in a zero water exchange system presenting higher survival and lowest FCR.

Boyd and Clay (2002) observed that bacterial flocs provide more stable

water quality. Because of the addition of molasses and the C: N ratio

adjustment, the bacterial community was able to use the dissolved nitrogen to

form biomass (Bartholdi and Rowdy, 2001; Ballester al., 2010). Hari et al.

(2006), reported that the addition of organic carbon to the water column led to

a significant increase in the biomass of the microbial community.

To stimulate the rapid uptake of ammonia by heterotrophic bacteria

labile organic carbon sources such as sucrose can be added to the culture water

(De Schryver et al., 2008). A carbon: nitrogen ratio (C: N) of system inputs

(feed and carbohydrates) above approximately 10 should result in efficient

ammonia assimilation (Ebeling et al., 2006). To effectively assimilate

ammonia, these bacteria must expand in biomass, however, the nitrogen they

assimilate is not taken out of the system unless the bacteria are removed.

4. Ammonia and nitrite dynamics

Ammonia and nitrite are generated during intensive aquaculture as a

consequence of aquatic animal excretion and microbial degradation of waste.

The presence of these compounds in water (Nootony et al.,2011) above 1.0 mg

N/L can cause adverse health effects in aquatic animals and create

environmental concerns if effluent is not properly treated (Timmons et al.,

2002 and Tchobanoglous et al., 2003).Many biological treatment systems have

been developed to maintain ammonia and nitrite concentrations in culture

water. Phytoplankton based systems are attractive because of their simplicity

and low operational cost but fail to sustain a stable operation because of

periodic phytoplankton bloom and crash cycles. Nitrifying biofilters have been

successfully employed in various aqua cultural applications. Despite many

advantages, the use of nitrifying bio filters remains costly.

Currently, biofloc technology systems have been receiving attention for

closed-water shrimp and tilapia cultivation because they feature high

production yield, water quality control, and feed protein recycling

simultaneously in the same culture unit (Crab et al. 2007 and Little et al.,

2008). In such system based on an enhancement of heterotrophic bacterial

growth to assimilate nitrogen into new cellular proteins (Schneider et al.,

2007).

As bacteria increase biomass, reaching a high density, tend to form

noticeable aggregates (bioflocs), which can be consumed by some cultured

animals as a natural food source (Buford et al., 2004; Schneider et al., 2006).

Biofloc technology is based on inorganic nitrogen assimilation into

heterotrophic bacterial biomass. Inorganic nitrogen controls in biofloc

technology systems can be accomplished by reducing feed protein contents to

increase organic carbon availability in water (Hargreaves, 2006 and Azim et

al., 2008).

Theoretically, TAN, nitrite, and nitrate concentrations in biofloc

technology systems should be stable and low as a result of inorganic nitrogen

conversion into bacterial biomass (Ebeling and Timmons, 2007; Schneider et

al., 2007). However, inorganic nitrogen profiles from both systems exhibited

the sequential accumulation of TAN followed by nitrite and nitrate (Nootony et

al., 2011). Such profiles are common during the start-up of biofilters in

recirculating aquaculture systems (Timmons et al., 2002; Hari et al., 2006;

Kuaka et al., 2009) and are characteristics of nitrification (Hargreaves, 1998;

Sauk et al., 2009; Xue et al., 2009).

Treatment tanks require approximately 6–7 weeks to establish the

complete nitrification. The lag in development of nitrification in treatment

tanks (i.e., using time for reaching peak ammonia as an indicator) can be

explained by competition for ammonia substrate between heterotrophs and

nitrifiers (Nootony et al., 2011) especially at substrate-limiting concentrations

(Malone et al., 2006). Organic carbon added to treatment tanks stimulated the

growth of heterotrophic bacteria, which have a growth rate that is about 10

times greater than nitrifiers that have doubling time of 26 hours for bacteria to

convert ammonia to nitrite (i.e. Nitrosamines) and 72 hours doubling time for

bacteria that convert nitrite to nitrate (i.e. Nitrobacter) (Tchobanoglous et al.,

2003; Hargreaves, 2006; Schneider et al., 2006).

Providing organic carbon (i.e., starch) favors heterotrophs and maintains

lower TAN concentration, thereby extending the acclimatization period

required for nitrifying bacteria activation, even under appropriate conditions for

nitrification. Supplying organic carbon to mediate nitrogen assimilating process

was beneficial because it reduced TAN and nitrite concentrations in water, and

this strategy might be employed as an alternative measure to lower the

unexpected increase of TAN and nitrite concentrations during system start-up.

Effective controls of TAN and nitrite concentrations commenced after

complete nitrification (Nootony et al., 2011) were established regardless of

organic carbon supplementation.

The role of nitrification in maintaining inorganic nitrogen concentrations

in control tanks was apparent as can be seen by the complete oxidation of TAN

and nitrite to nitrate for the remainder of the experiment. The complete

oxidation of TAN and nitrite by nitrifiers in treatment tanks receiving organic

carbon addition was favored by high oxygen concentration, suitable alkalinity,

and bioflocs, and tank walls likely served as attachment sites for slow growing

nitrifying bacteria.(Nootony et al., 2011).

Significant nitrifying activity in biofloc systems was also described by

Azim and Little (2008). In that work, nitrate concentration in tilapia tanks

reached 250 mg N/L after 11weeks despite the tanks being seeded with 350

mg/L of bioflocs and fed daily with low-protein (24%) diets. Assimilation and

nitrification occur simultaneously in many activated sludge process units that

treat domestic and industrial wastewater (Charley et al., 1980; Tchobanoglous

et al., 2003).

Thakur and Lin (2003) reported nitrogen loss as high as 36% during

zero-discharged shrimp cultivation in concrete tanks. Nitrogen loss as high as

55% was reported in brackish ponds with very limited water discharge (Daniels

and Boyd, 1989). Denitrification and ammonia volatilization were assumed to

be the pathways for nitrogen loss (Thakur and Lin, 2003; Hari et al., 2006).

Ammonia volatilization was not expected to be significant because TAN was

less than 1.0 mg N/L and the pH was between 7 and 8 so that the major fraction

of TAN was in the soluble ionized form (i.e., NH4+-N).

Nitrogen loss via denitrification was more likely because of the high

level of nitrate in water, the availability of dissolved organic carbon, and the

presence of anaerobic pockets at the inner region of bioflocs or caused by

bioflocs domination on tank bottoms (Nootony et al., 2011). Further study is

necessary to identify ecological relationships between nitrifying and

heterotrophic bacteria in biofloc systems. The Addition of organic carbon may

be conducted until the establishment of complete nitrification or used as a

strategy to quickly reduce TAN and nitrite concentrations. (Nootony et al.,

2011).

The use of sodium carbonate, as proposed by Furtado et al. (2011),

helped to keep the pH values suitable for good growth performance of the

species (Van Wyk and Scarpa 1999).

Kuhn et al. (2010) demonstrated that chronic exposure of juvenile L.

vannamei to nitrate levels up to 435 mg NO3-N L−1(1927 mg NO3L−1) over six

weeks did not affect growth and survival but did have a negative impact on

shrimp biomass and antennae length, with greater impact at lower salinities (2–

18 ppt). Mean TAN and NO2-N concentrations were low despite the high feed

rates presumptively due to phytoplankton uptake and nitrification. Mean NO3-

N, settleable solids, total suspended solids, and total volatile solids

concentrations were comparable to values reported for freshwater (Green,

2010) and brackish water/marine (Ray etal. 2010a; Vinita et al., 2010).

The Biofloc technology is based on the manipulation of microbial

community through the addition of a carbon source that promotes the

development of heterotrophic bacteria (Souza et al., 2012). These bacteria use

the organic carbon and the inorganic nitrogen present in the water to produce

their biomass through by removing toxic ammonia from the culture system

(Hargreaves, 2006; Schryver et al., 2008). The basic principle of the BFT

system is the retention of waste and its conversion into biofloc as a natural food

source within the culture system (Azim and little 2008).

One of the benefits of this system is the bacterial uptake of nitrogen,

including ammonia (Burford et al., 2003), and its conversion into cellular

protein, which also provides supplemental source of nutrition (McIntosh ,

2000; Burford et al., 2004b; Wasielesky et al., 2006) and possibly reducing

the demand for protein in feed (Burford et al., 2003; Ballester et al., 2010)

In aquaculture systems, phytoplankton and bacteria play crucial role in

the processing of nitrogenous wastes (Shiloh and Ramon, 1982). According to

Boyd and Clay (2002), the water quality of a heterotrophic microbial-based

production system containing bacterial flocs is more stable than that of a

phytoplankton-based production system.

The concentrations of ammonia and nitrite in molasses decreased faster

than control because this microbial community was able to utilize the nitrogen

contributing to the maintenance of water quality (Souza et al., 2012).

Moreover, the improvement in shrimp performance observed in molasses can

be a result of supplemental food source of flocs available in the system (Souza

et al., 2012). Several works have reported the benefits of microorganisms as

food source (Ballester et al., 2007). The microorganisms on the biofilm served

as complementary food source providing nutritional benefits for shrimp,

improving survival and biomass (Souza et al., 2012).

However, apart from serving as a direct source of nutrients to shrimp,

there is evidence that the microorganisms present in the flocs also exert

positive effect on shrimp digestive enzyme activity and gut microflora. Souza t

al. (2012) demonstrated that molasses can be used as a tool to prevent

increases in the TAN and nitrite concentrations during the nursery phase of F.

brasiliensis culture under conditions of limited water discharge. The shrimp

performance results suggest that the microbial community served as a

complementary food and improved the rearing conditions and the shrimp

growth and survival. The concentrations of TAN and nitrite recorded

throughout the trial was maintained in adequate levels recommended for

juveniles of Pacific white shrimp (Lin and Chen, 2001, 2003). The low

concentrations of nitrite observed during the culture period suggest the

complete oxidation of ammonia to nitrate (Cohen et al., 2005).

Studies evaluating water quality in zero-exchange system report low

concentrations of ammonia and nitrite (Burford et al., 2004; McIntosh et al.,

2000; Ray et al., 2010; Vinita et al., 2010; Wasielesky et al., 2006), resulting

from the removal of these compounds by microbial community (Ebeling et al.,

2006). Nitrate concentration was low, due to the lower concentration of

ammonia nitrogen available to the oxidation by nitrifying bacteria (Holl et al.,

2011). The absorption of this reduced form of inorganic nitrogen by

phytoplankton was probably the primary cause, since the Chl a concentrations

in this treatment was higher than the others treatments. The ammonia nitrogen

was found as the preferred source of inorganic nitrogen for phytoplankton in

intensive biofloc shrimp culture systems as evidenced by Holl et al., (2011).

The use of carbon sources in intensive systems promotes succession

and dominance of bacteria over microalgae (González-Félix et al., 2007; Ju et

al., 2008a, b).

However even with the application of organic carbon at a C: N ratio of 20:1,

fluctuations occurred in TAN concentrations (Brito et al.,2013). Thakur and

Lin (2003), Cohen et al.(2005), Azim and Little (2008), and Ray et al. (2010)

reported similar fluctuations in TAN concentration with an addition of organic

carbon. This variation was probably related to the state of maturation of the

system and the amount of these nutrients used by the microalgae and by

nitrifying and heterotrophic bacteria (Hargreaves, 1998, 2006). The higher

initial TAN concentrations in treatments without biofloc caused an acceleration

of the nitrification process, with a consequent pH and alkalinity reduction

(Brito et al., 2013).

Bacteria require substantial amounts of oxygen to assimilate ammonia

and nitrifying bacteria can be slow to establish, resulting in spikes of toxic

ammonia and nitrite (Ray et al., 2009). In closed aquaculture systems NO3–N

may accumulate to concentrations that can inhibit shrimp survival and growth

(Kuhn et al., 2010). Given the proper conditions, anaerobic denitrifying

bacteria can convert NO3–N or NO2–N to nontoxic N2 gas which is then

released into the atmosphere (Hamlin et al., 2008). These bacteria are also

capable of assimilating PO4 and they typically generate alkalinity (Ray et al.,

2011).

However, in suboptimal anaerobic conditions some bacteria can reduce

NO3–N back to the toxic TAN compound through the nitrate reduction to

ammonia process (Van Rijn et al., 2006). Ray et al. (2010a) documented a

significant reduction in NO3–N and significant increase of alkalinity in systems

with settling chambers compared to those without. The results of their study

indicate that these simple filtration systems can not only remove particles, but

may also serve as denitrification chambers (Ray et al .,2011). This dual

function may make settling chambers an attractive. Higher salinity can help

protect animals from high concentrations of toxic nitrogen compounds, which

can be problematic in intensive culture systems (Ray et al., 2011)

5. Growth and feed performance

The high levels of survival were related to the high water quality that

was maintained during the study (Wasielesky et al., 2013) and also to the

availability of in situ production of additional feed provided by the bioflocs

(Crab et al., 2012) The natural food source provided by the growth of the

microbial community offers a high-quality food supplement that benefits the

shrimp, especially during the nursery phase (Emerenciano et al., 2012).

Because of the improved utilization of natural productivity, a decreased FCR

was expected (Wasielesky et al., 2006 ; Mishra et al., 2008). The high survival

in all treatments at the end of the experiment was also a good indication that

the bioflocs contributed to the health of the shrimp even after exposure to high

densities.

Crab et al. (2012) stated that bacteria and their products may have

immune stimulatory effects on animal growth. This effect can increase the

survival and resistance of animals, even during stressful situations (Wasielesky

et al., 2013). Using greenhouse-enclosed limited-exchange systems can also be

beneficial for shrimp nursery production in temperate climate areas to

accommodate PL early season stocking (“head start”), when the ambient water

temperature in grow-out ponds is too low for the shrimp to survive and/or grow

(Correie et al., 2014). This practice can extend the grow-out season to produce

larger shrimp or to grow multiple crops per year (Samocha et al., 2000a, b;

Samocha and Benner, 2001; McCabe et al., 2003).

There is a need to develop diets for shrimp cultured in limited exchange

nursery systems that will provide sufficient protein for shrimp production while

minimizing the amount of nitrogen being introduced into the culture medium

(McIntosh et al., 2001). Shrimp typically have a higher dietary protein

requirement during the nursery phase than at later stages (Velasco et al., 2000).

Emerenciano et al. (2012) presented higher levels of final weight, final biomass

and weight gain of Brasiliensis reared in BFT treatments compared with clear

water. Moreover, these authors confirmed favorable nutritional quality of

biofloc-enhancing shrimp performance. Therefore, it is reasonable to assume

that the addition of carbon led to microbial community with proprieties that

contributed to shrimp performance (Souza et al., 2012).

Shrimp performance reared in the environment with molasses addition

exhibited a better survival rate, final weight and SGR significantly higher than

those of the control. These results are in agreement with Krummenauer (2008),

who demonstrated the efficacy of the BFT culture system in high-intensity

shrimp culture with a production above 2.5 kg/m2. Other authors (Otoshi et al.,

2006; Otoshi, et al., 2007 a) have reported production values ranging from 4.5

to 10 kg/m2, confirming the success of this system for shrimp production

.Based on final growth indicators, the addition of molasses had no effect on

shrimp under conditions of limited water discharge (Samocha et al., 2007).

These findings demonstrated that this system has no negative effect on shrimp

(Souza et al., 2012).

Biofloc technology makes it possible to minimize water exchange and

water usage in aquaculture systems through maintaining adequate water quality

within the culture unit ( Crab et al.,2012), while producing low cost bioflocs

rich in protein, which in turn can serve as a feed for aquatic organisms (Crab,

2010 ; Crab et al., 2007, 2009). Compared to conventional water treatment

technologies used in aquaculture, Biofloc technology provides a more

economical alternative (Decrease of water treatment expenses in the order of

30%), and additionally, a potential gain on feed expenses (the efficiency of

protein utilization is twice as high in biofloc technology systems when

compared to conventional ponds), making it a low-cost sustainable constituent

to future aquaculture development (De Schryver et al., 2008).

On the other hand, several factors promoted the implementation of the

technique. Firstly, water has become scarce or expensive to an extent of

limiting aquaculture development. Secondly, the release of polluted effluents

into the environment is prohibited in most countries. Thirdly, severe outbreaks

of infectious diseases led to more stringent biosecurity measures, such as

reducing water exchange rates (Crab et al., 2012).

Kuhn et al. (2009) included dried and processed bioflocs from tilapia

ponds into shrimp feed and obtained about 1.6 times higher average weight

gain per week than that obtained with commercial diets. At the University of

the Virgin Islands, researchers are currently looking at tilapia and shrimp

polyculture in intensive bacterial-based, aerated tanks. Thermal trophic

approach of combining species with different specific feeding niches brings

about a more complete use of resources than in the monoculture approach

(Rahman et al., 2008). With biofloc technology where nitrogenous waste

generated by the cultivated organisms is converted into bacterial biomass

(containing protein), in situ feed production is stimulated through the addition

of an external carbon aource (Schneider et al., 2005).

The potential feed gain of the application of biofloc technology is

estimated to be in the order of 10–20% (Schryver et al., 2008). With this,

production costs will decline considerably since food represents 40-50% of the

total production costs (Craig and Aelfric, 2002). By eliminating the dependence

on sunlight, These systems can be housed in the controlled environment of

insulated buildings, leading to a reduction in energy costs during the cold

months (Ray et al., 2009). It has been suggested that natural productivity in

zero-exchange shrimp production systems provide supplemental food

resources, reducing feed costs and improving shrimp growth rate (Otoshi et al.,

2011; Wasielesky et al., 2006). Ray et al. (2009) also reported shrimp

production 17% higher and FCR 18% lower in photoautotrophic raceway,

compared to a totally heterotrophic raceway, and suggested that

photoautotrophic organisms may have provided supplementary feed for the

shrimp. Feed conversion ratio is an important parameter in aquaculture because

feed costs generally represent up to 60% of the total production cost (Cuzon et

al., 2004 and et al., 2002).

6. Health state

Besides providing supplemental nutrition, like protein, lipid, mineral

and vitamin (Izquierdo et al., 2006; Ju et al., 2008b; Moss et al., 2006 ; Xu et

al., 2012b), the bioflocs is a source of abundant natural microbes and bioactive

compounds that could exert a positive effect on the physiological health of

cultured shrimp. Xu and Pan (2013) indicated that the presence and digestion

of the biofloc ingested by the shrimp may release substances in the

gastrointestinal tract that could potentially stimulate the innate immune

response (especially phagocytosis). Xu and Pan (2013) reported that it is

possible that some kinds of beneficial bacteria such as Bacillus sp. in the

ingested biofloc could facilitate the modification of physiological and

immunological status of the host through the colonization in the

gastrointestinal tract and the induction of changes in the endogenous

microbiota (Johnson et al., 2008; Li et al., 2009). Moreover, it should be noted

that both microbial components (e.g. Polysaccharides) and bioactive

compounds (e.g. carotenoids) existing in the biofloc (Ju et al., 2008a) could

exert an immune-stimulating effect and this action was continuous as long as

shrimp consumed biofloc.

However, the modes of action of biofloc on innate immune system of

shrimp are complicated and unknown at present. Xu and Pan (2013) assumed

that based on its composition characteristics, the biofloc may (i) play a role in

antioxidant activity because it contains an appropriate amount of antioxidants

such as carotenoids and fat-soluble vitamins (Ju et al., 2008a) and (ii) stimulate

digestive enzyme activities and improve feed utilization (Xu and Pan, 2012 ;

Xu et al., 2012a), thereby increasing the assimilation of dietary antioxidants

from the feed ( Xu and Pan 2013). From this point of view, developing biofloc

in the culture system is a promising management strategy for the improvement

of physiological health of cultured shrimp. Further investigations are needed to

verify the beneficial effects of biofloc serving as potential sources of immuno-

stimulants and antioxidants on physiological responses and healthy culture of

shrimp, and on how to manipulate microbial communities and active

compounds of biofloc under different culture conditions (Xu and Pan 2013).

Environmental damage associated with effluent discharge and massive

crop losses due to disease outbreaks have created a need for more sustainable

and bio secure shrimp production practices (Cowey and Cho, 1991; Samocha,

2009). Implementation of limited or no water exchange shrimp production

systems has the potential to minimize these negative environmental impacts

and disease outbreaks, while conserving water resources and not compromising

profit. (Correia et al., 2014).

Survival rates also improved not only because of a better nutrition

(Burford et al., 2004), but also due to a stable bacterial community able to

control pathogenic outbreak (Thompson et al., 1999). Furthermore, it has been

suggested that floc benefits the shrimp immune system (Hsieh et al., 2007),

since bacteria isolated from FLOC produced carotenoids, retinoid, poly-β-

hydroxybutyrate (Defoirdt et al., 2007; Nhan et al., 2010) and exoenzymes

(Bairagi et al., 2002).

Aguilera-Rivera et al. (2014) revealed the presence of a unique group

of vibrio exclusively found in FLOC, and shrimp showed better health status. It

suggests not only the strengthening of the shrimp immune system by the

molecules described above, but also a structuring of the microbial community

that may be keeping in equilibrium and prevent an outbreak from unidentified

opportunistic pathogenic Vibrio.

The differences in vibrio species observed between the control and

FLOC resided in the fact that some microorganisms in FLOC particles play a

key role on bacterial communities. Histopathology showed that shrimp in

FLOC had better immune status than in CW, where more lesions occurred with

or without probiotic. Therefore, although probiotic affected survival by

stabilizing to some extent the shrimp digestive flora, this was not enough to

enhance growth rate (Aguilera- Rivera et al., 2014).

7. Nutritional value of biofloc

Moreover, the biofloc derived from shrimp culture water is rich in

various bioactive compounds including carotenoids chlorophylls,

polysaccharides, phytosterols, taurine and fat-soluble vitamins (Ju et al.,

2008a), all of which can contribute to a healty status of cultured shrimp (Xu

and Pan, 2013). Apart from being a source of quality proteins, biofloc are rich

source of growth promoters and bioactive compounds (Ju et al., 2008a) which

enhance digestive enzymes (Xu and Pan, 2012) and health status of the

cultured shrimps (Singh et al., 2005).

Total yield of biofloc per production cycle was 4.03 kilograms wheat

flour was used to produce 1 kg of bioflocs (ShyneAnand et al., 2014).

Proximate composition (%) of biofloc : the dried biofloc contained 24.30%

crude protein, 3.53 % crude lipid and 29.24% nitrogen free extract (NFE).The

mean ash content and acid insoluble ash content was 31.98 and 10.75% of

dried biofloc respectively (Shyne Anand et al., 2014). Recently it has been

reported that use of biofloc as a dietary ingredient in shrimp diet enhances the

growth rate of L. vannamei (Kuhn et al., 2009, 2010).

Ballester et al. (2010) reported that biofloc is composed of attached

heterotrophic bacteria, filamentous cyanobacteria, dinoflagellates, ciliates,

flagellates and rotifers. Ju et al. (2008a) reported the dominance of algal

communities over bacterial biomass in flocs collected from outdoor shrimp

culture units. Proximate analysis of the biofloc in was in agreement with the

findings of Ballester et al. (2010) who reported 30.4% crude protein (CP) with

wheat flour and molasses as carbohydrate sources. Ju et al. (2008a) reported

that chlorophyll-dominated biofloc contained higher crude protein content

(42%) than flocs dominated by diatoms (26-34%) and bacteria (38%). This

further suggests that the microbiota that constitutes the biofloc is likely to

affect the protein content of the bioflocs (Shyne Anand et al., 2014).

It has been documented that bioflocs are the rich source of many

bioactive compounds such as carotenoids, chlorophylls, phytosterols,

bromophenols, amino sugars (Ju et al.,2008a) and anti-bacterial compounds

(Crab et al., 2010). This suggests that microbial components, unknown growth

factors or probiotic microorganisms like Bacillus, Lactobacillus present in the

biofloc might have resulted in significantly higher growth rate and better FCR

in shrimp fed with biofloc incorporated diet (Shyne Anand et al., 2014).

Kuhn et al. (2010) replaced the fish meal by biofloc in L. vannamei diet

and recorded significantly higher growth rate at 10 and 15%, and non-

significant difference at 21 and 30% dietary inclusion level of biofloc. The

findings of Shyne Anand et al.(2014) agree in general with those of Wang

(2007) and Anand et al.(2013b) who reported that the increase in dietary

supplementation of probiotic or epiphytic algae in shrimp diet does not increase

proportionately the digestive enzyme activities and growth of shrimp.

Moreover, reduction in growth rate of fishes was recorded at higher

level of microbial supplementation (Ajiboye et al., 2012; Kiessling and

Askbrandit, 1993) as microbial products at higher level tend to reduce the feed

palatability and digestibility (Kiessling and Askbrandit, 1993). Seeing the cost-

effectiveness, the inclusion of biofloc at 4% level is beneficial in improving

growth performance and digestive enzyme activities in shrimp (Shyne Anand et

al., 2014). The study demonstrated that dietary supplementation of biofloc at

4–8% level had beneficial effects on growth performance and digestive enzyme

activities in monodon.

Current method of biofloc production using ammonium sulphate as

nitrogen and wheat flour as carbon source is cheaper and easier compared to

bioreactors. These findings may encourage feed manufacturers to consider

biofloc as a viable alternative dietary supplement (Shyne Anand et al., 2014).

Although bioflocs meet nutritional standards to serve as an aquaculture feed in

general, research has shown that the capacity of the technique to control the

water quality in the culture system and the nutritional properties of the flocs are

influenced by the type of carbon source used to produce the flocs (Crab, 2010).

Different organic carbon sources each stimulated specific bacteria,

protozoa and algae, and hence influenced the microbial composition and

community organization of the bioflocs and thereby their nutritional properties

(Crab, 2010). Feeding experiment revealed that besides these characteristics,

the type of carbon source also influenced the availability, palatability and

digestibility for the cultured organisms (Crab, 2010). Overall, bioflocs

produced on glycerol gave the best results in previous work. However, further

research should focus on the use of low-cost non-conventional agro-industrial

residues as carbon source and hence upgrade waste to nutritious feed. Different

carbon sources will stimulate the growth of the indigenous microbiota in

another way and thus exert a distinctive effect on water quality, in situ feed

(Crab et al., 2012).

Much of the nitrogen input in culture systems enters the water column as

total ammonia-nitrogen generated by feed which is not converted into shrimp

tissue. (Correia et al., 2014). Thakur and Lin (2003) showed that under no

exchange Penaeus monodon assimilated only 23–31%of the nitrogen added to

the system. The presence of microbial and algal communities in limited

discharge systems helps with the recycling of the system’s metabolites

(Burford al., 2003; Wang, 2003). Besides the nutrient recycling aspect, the

dense bacterial community that develops in such systems play a significant role

in the production of single cell microbial protein (“biofloc”) that can provide

supplemental natural feed for the shrimp ( Rowdy et al., 2001).

Wasielesky et al. (2006) suggested that this enhanced natural production

in zero exchange production systems and allows the use of low protein feeds

with no adverse effect on shrimp performance compared to high protein feeds.

Several studies show that bioflocs contain not only considerable level of

protein but also HUFA (Ekasari et al. ,2010) and vitamin C (Crab et al. , 2012)

which are required to support gonadal maturation and high egg quantity and

quality (Dabrowski and Ciereszko, 2001).

It has been suggested that continually cropping out a portion of the

microbial community produces a younger, healthier community that may

thereby provide enhanced nutritional benefits to culture animals (Turker et al.,

2003). Further research is needed to fully understand the ways that solids

management affects shrimp growth rate.

FLOC was defined as a medium rich in organic matter, made of

particulate biomass and colonized by bacteria (Aguilera-Rivera et al.,

2014). From a nutritional point of view it helps shrimp to gain weight

owing to an abundance of native protein sources from protozoa,

filamentous bacteria, nematodes, ciliates, flagellates, and rotifers

(Decamp et al., 2002; Ray et al., 2010).

8. Protein content in diet

The increase in biofloc are provided by the development of

heterotrophic bacteria which feed on ammonia and organic carbon from

molasses and the autotrophic bacteria that consume nitrogen compounds (e.g.,

ammonia and nitrite), reducing their concentration in the water while

consuming inorganic carbon (e.g., alkalinity) (Correia et al., 2014). This

biofloc, formed by the bacterial biomass and other microorganisms present in

the medium, has substantial nutritional value that contributed to the diet of the

cultured shrimp, providing a feed supplement (Correia et al.2014).

Correia et al. (2014) suggested that substituting high (40%) with low-

protein (30%) feed in the shrimp nursery phase in a biofloc dominated system

may provide an alternative to improve biofloc technology. Shrimp fed the high

protein diet had significantly higher SGR and final weight than those fed the

low protein diet, but there were no significant differences in shrimp survival or

PER between the two diets (Correia et al.,2014).

Several advantages to using the low protein feed can be shown. Firstly

better water quality, as nitrite, nitrate and phosphate were lower , second, feed

with lower protein content is cheaper , and third , the use of lower protein feed

in this biofloc system can reduce the environmental impacts from shrimp

culture, through lower protein use and water exchange requirement (Correia et

al., 2014).

Biofloc technology is a new concept in aquaculture, where

manipulation of the microbial community is carried out under controlled

conditions within the culture system with the raised animals (De Schryver et

al., 2008). This system facilitates the production of aquatic animals at high

stocking densities in a sustainable and bio-secure fashion (McCabe et al., 2003;

McNeil, 2000; Vinita et al., 2009). In some cases the protein content of feed

can be reduced due to partial protein supplementation by the microbial

community (Burford et al., 2004; Wasielesky et al., 2006).

One of the advantages of operating bacterial-driven system versus a

conventional phytoplankton dominated pond is that microbial production is

limited by the availability of organic matter or substrate rather than light

(Beloit al .,2013), giving rise to the potential for this system in indoor

conditions (Azim et al., 2008).

9. Water quality parameters

The constant mixing and high turbulence of the water provided in the

biofloc tanks may have inhibited the formation of blooms of relatively larger

planktonic, filamentous species (Schrader et al ., 2011) that can commonly

occur and dominate phytoplankton communities in catfish productions ponds

(Vander Ploeg and Tucker, 1993). In addition, the high turbidity of the water in

the tanks may have reduced suitable conditions for the growth of planktonic,

filamentous cyanobacteria due to the reduced availability of photo synthetically

active radiation (Schrader et al., 2011).

The BFT systems used to culture channel catfish favored the

development of phytoplankton communities dominated by small colonial types

of cyanobacteria and small fast-growing unicellular or small colonial types of

green algae and diatoms (Schrader et al., 2011). Water quality deterioration on

the other hand may cause stress and eventually affect the growth and

reproductive performance of the fish (Brune, 2000).

Hydrated lime (Ca (OH)2) was used to maintain alkalinity and pH

above 100 mg L-1 and 7.5, respectively in the biofloc tank, a pH reduction

generally occurs (Wasielesky et al., 2006; Emerenciano et al., 2011) due to

alkalinity consumption during ammonia–nitrogen conversion processes

(Ebeling et al., 2006). According to Furtado et al. (2011), levels under 100 mg

L-1 of CaCO3 and pH 7 for prolonged periods of time can affect the growth

performance of shrimp in biofloc. The addition of inorganic carbon was

required to maintain desirable levels during the nitrite production process,

(Briton et al., 2013) which consumed calcium carbonate, releasing CO2 and

hydrogen into the water (Hargreaves 1998).

10. Aeration in biofloc

In general, most research makes use of in situ developed microbial floc

for growth performance of shrimp (Hari et al., 2006; Xu and Pan, 2012).

However, these in situ based techniques need additional oxygen demands for

microbial respiration, in addition to the oxygen demand of shrimp (Burford et

al., 2003; Tacon et al., 2002). This added oxygen demand requires additional

aerators, which increases the aeration expenses in shrimp farms compared to

conventional shrimp culture systems (Tacon et al., 2002).

Moreover, increased production of microbial floc particle is also a

matter of challenge if production exceeds consumption by shrimp

(ShyneAnand et al., 2014).

11. Suspended solids

Generally TAN and nitrite concentrations Less than 1.0 mg N/L are

recommended for long term exposure (Timmons et al., 2002). Severe growth

inhibition and increased mortality of tilapia were observed when the suspended

solids concentration exceeds 850 mg/L (Little et al., 2008). Thus, maintaining

optimal suspended solids concentration may be the critical aspect in managing

biofloc technology systems, with the recommended maximum suspended solids

concentration at 500 mg/L (Azim and Little, 2008; Little et al., 2008).

Removal of the solids was not conducted in their study, resulting in

excessive suspended solids concentration (i.e., >500 mg/L) that may have

reduced visibility and consequently the ability of tilapia to find feed (Azim and

Little 2008). Biofloc technology is not yet fully predictable and can therefore

be risky to implement at farm level. Possible monitoring tools are the

concentration of total suspended solids or bioflocs, and the settling ability of

the biofloc which can both be measured quickly and easily (De Schryver et al.,

2008).

Using 6200-L outdoor tanks, half with simple settling chambers and half

without, Ray et al. (2010a) demonstrated that managing biofloc concentration

could significantly improve shrimp growth rate, FCR, and biomass production.

Also, the authors showed that settling chambers contributed significantly to

decreased nitrate and phosphate concentrations and significantly increased

alkalinity concentration in the shrimp culture systems.